【摘要】 目的:用真核表达siRNA干扰技术特异性抑制宫颈癌细胞CaSKi中HPV16E7癌基因的表达。方法:运用DNA重组技术构建表达特异性siRNA的pGenesil-1/E7(PS7)重组质粒,用脂质体分别将重组质粒转染宫颈癌细胞系CaSKi细胞内,通过半定量RT-PCR实验检测CaSKi/PS7细胞中E7mRNA水平的变化;再用western blotting检测CaSKi/PS7细胞中E7蛋白、磷酸化pRb和去磷酸化Rb的表达;用免疫细胞化学法检测细胞中ki-67、NF-κB、bcl-2和p16的表达。结果:成功构建了真核细胞表达的、特异性siRNA的PSN和PS7重组质粒。在RNA干扰24、48和72小时后CaSKi/PS7细胞中E7mRNA分别下降了21.05%、51.80%和49.10%;RNA干扰后的CaSKi/PS7细胞中E7蛋白的表达逐渐减低,而且磷酸化pRb表达降低和去磷酸化Rb的表达升高;与CaSKi/PSN细胞相比,CaSKi/PS7细胞中的ki-67、NF-κB和bcl-2表达降低,而p16的表达增加。结论:本研究成功构建了真核细胞体内表达特异性siRNA重组体,有效地抑制了宫颈癌细胞CaSKi中HPV16E7基因的表达,为RNA干扰应用于研究宫颈癌发病分子机制提供了新的资料和方法。更多还原

【Abstract】 Objective:To specifically inhibit the expression of oncogene E7 of HPV16 in CaSKi cervical carcinoma cell by RNA interference.Methods:The recombinant plasmids which expressed small hairpin RNA(shRNA) in vivo to target E7 gene was constructed.By use of lipofectamine2000 the plasmids were transfeced into cervical carcinoma cell CaSKi.E7mRNA were detected by semi-quantitative RT-PCR and Rb,E7 were detected by western blotting and other proteins associated to cell profilation such as ki-67,NF-κB,p16 and bcl-2 by immunocytochemistry method.Results:The plasmids pGENESIL-1/Negative(PSN) and pGENESIL-1/E7(PS7) were successfully constructed.The amount of E7mRNA in CaSKi/PS7 was decreased 21.05%,51.80% and 49.10% respctively after 24h,48h and 72h.In CaSKi/PS7 the expression of hypo-phosphorylated Rb was increased but that of hyper-phosphorylated Rb was decreased.The expression of ki-67,NF-κB and bcl-2 in CaSKi/PS7 were decreased but that of p16 was increased.Conclusion:These findings suggested that inhibition of the expression of oncogene E7 in HPV16 by means of RNA interference was effective and provided new materials for prevention and treatment of cervical carcinoma infected by HPV16.更多还原